Assay and other reactions involving droplets

Abstract

The present invention generally relates to droplets and/or emulsions, such as multiple emulsions. In some cases, the droplets and/or emulsions may be used in assays, and in certain embodiments, the droplet or emulsion may be hardened to form a gel. In some aspects, a heterogeneous assay can be performed using a gel. For example, a droplet may be hardened to form a gel, where the droplet contains a cell, DNA, or other suitable species. The gel may be exposed to a reactant, and the reactant may interact with the gel and/or with the cell, DNA, etc., in some fashion. For example, the reactant may diffuse through the gel, or the hardened particle may liquefy to form a liquid state, allowing the reactant to interact with the cell. As a specific example, DNA contained within a gel particle may be subjected to PCR (polymerase chain reaction) amplification, e.g., by using PCR primers able to bind to the gel as it forms. As the DNA is amplified using PCR, some of the DNA will be bound to the gel via the PCR primer. After the PCR reaction, unbound DNA may be removed from the gel, e.g., via diffusion or washing. Thus, a gel particle having bound DNA may be formed in one embodiment of the invention.

What is claimed is: 
     
       1. A method of polynucleotide amplification comprising:
 a. generating a library of droplets comprising a plurality of polynucleotide primers, 
 wherein the library comprises more than 10 5  different polynucleotide primers, wherein some of the droplets in the library are distinct, and wherein the library of droplets excludes polynucleotides to be amplified; 
 b. generating a set of droplets, wherein the droplets comprise at least one polynucleotide to be amplified; 
 c. merging a first droplet from the library of droplets with a second droplet from the set of droplets to form a coalesced droplet; and 
 d. performing an amplification reaction on at least one polynucleotide in the coalesced droplet to form at least one amplicon. 
 
     
     
       2. The method of  claim 1 , wherein the library of droplets comprises more than 10 6  polynucleotide primers. 
     
     
       3. The method of  claim 1 , wherein the amplification reaction is whole genome amplification. 
     
     
       4. The method of  claim 1 , further comprising sequencing the at least one amplicon. 
     
     
       5. The method of  claim 1 , wherein the amplification reaction is selected from the group consisting of: Polymerase Chain Reaction (PCR), reverse-transcription PCR and real-time PCR. 
     
     
       6. The method of  claim 1 , wherein the amplification reaction is reverse transcription PCR. 
     
     
       7. The method of  claim 1 , wherein the first droplet further comprises amplification reagents. 
     
     
       8. The method of  claim 1 , wherein the second droplet further comprises amplification reagents. 
     
     
       9. The method of  claim 1 , wherein the at least one polynucleotide to be amplified comprises a polymorphism. 
     
     
       10. The method of  claim 1 , further comprising forming a plurality of coalesced droplets at a rate of at least 10 droplets per second. 
     
     
       11. The method of  claim 1 , wherein the average diameter of the first, second or coalesced droplet is at least 1 micrometer. 
     
     
       12. The method of  claim 1 , wherein the library of droplets comprises aqueous droplets within an oil phase. 
     
     
       13. The method of  claim 1 , wherein the set of droplets comprises aqueous droplets within an oil phase. 
     
     
       14. The method of  claim 1 , wherein the polynucleotide primers comprise an acrydite moiety. 
     
     
       15. The method of  claim 1 , further comprising hardening the coalesced droplet to form a gel. 
     
     
       16. The method of  claim 15 , wherein the polynucleotide primers are bound to the gel. 
     
     
       17. The method of  claim 15 , wherein the coalesced droplet comprises a gelation initiator. 
     
     
       18. The method of  claim 17 , wherein the gelation initiator co-flows with an aqueous phase. 
     
     
       19. The method of  claim 17 , wherein the gelation initiator co-flows with an oil phase. 
     
     
       20. The method of  claim 17 , wherein the gelation initiator is selected from the group consisting of ammonium persulfate, TEMED and Ca 2+ . 
     
     
       21. The method of  claim 15 , wherein the gel is poly(acrylamide). 
     
     
       22. The method of  claim 1 , wherein the merging of the first droplet with the second droplet is controlled. 
     
     
       23. The method of  claim 22 , wherein the merging of the first droplet with the second droplet is electrically controlled. 
     
     
       24. The method of  claim 1 , wherein the amplification is performed using a primer from the library of primers. 
     
     
       25. The method of  claim 17 , wherein the gelation initiator comprises a reducing agent. 
     
     
       26. The method of  claim 1 , wherein the droplets comprise an identifier. 
     
     
       27. The method of  claim 1 , wherein droplets within the library of droplets comprise different identifiers. 
     
     
       28. The method of  claim 26 , wherein the identifier is an optical identifier. 
     
     
       29. The method of  claim 26 , wherein the identifier is a fluorophore. 
     
     
       30. The method of  claim 13 , wherein the oil phase comprises fluorocarbon oil. 
     
     
       31. The method of  claim 30 , wherein the fluorocarbon oil comprises Krytox. 
     
     
       32. The method of  claim 1 , wherein the droplets comprise a fluoro surfactant. 
     
     
       33. The method of  claim 1 , wherein the droplets comprise an ammonium carboxylate salt surfactant. 
     
     
       34. The method of  claim 1 , wherein the droplets comprise a surfactant selected from the group consisting of Span80, Span80/Tween-20, Span80/Triton X-100, Abil EM90, Abil we09, polyglycerol polyricinoleate “PGPR90”, Tween-85, 749 Fluid, and ammonium carboxylate salt of Krytox.